Inhibition of a-Ketoglutarate Dehydrogenase due to Arsenite Exposure

Erik Bergquist

Department of Biology, Department of Chemistry, The University of Montana, Missoula, Montana 59801

Abstract

a-Ketoglutarate dehydrogenase (KGDH) was inhibited with increasing arsenite exposure. Activity loss was linked to modification of the sulfhydryl groups on lipoic acid, the essential cofactor of the KGDH E2 subunit. Modification of lipoic acid by arsenite was found to be irreversible causing 25% enzyme inhibition as low as 250ppb As(III). To verify lipoic acid modification, anti-lipoic acid antibodies and mass spectroscopy were performed enabling the confirmation of a mechanism for arsenite induced oxidative stress.

Introduction

Oxidative stress is the formation of reactive oxygen species that exceed the cells natural antioxidant capabilities. Arsenic is present in surface and ground water as arsenate, As(V), and arsenite, As(III), with As(III) the more toxic oxidation state. The toxicity and carcinogenicity of arsenite have been ascribed to its ability to induce oxidative stress. Human exposure to arsenite occurs naturally through contaminated drinking water. Chronic arsenite exposure has been linked to cancer of the lung, skin, kidney, urinary bladder, and liver.[i] A number of mitochondrial enzymes contain the putative arsenite reactive lipoic acid as an essential cofactor. The enzyme a-ketoglutarate dehydrogenase (KGDH) is one of these lipoic acid containing enzymes and the basis of this research.

KGDH is a three-enzyme complex. Each enzyme requires an essential cofactor; a-ketoglutarate dehydrogenase requires thiamine pyrophosphate (TPP), transuccinylase requires lipoic acid, and dihydrolipoyl dehydrogenase requires flavin adenine dinucleotide (FAD). KGDH is the rate-determining enzyme of the citric acid cycle, catalyzing the reaction of isocitrate to succinyl-CoA. In addition, KGDH creates metabolic precursor molecules as well as reducing power in the form of NADH for the first step of the electron transport chain. Thus loss of KGDH activity causes a halt of the citric acid cycle, formation of metabolic precursor molecules, and flow of electrons through the electron transport chain.

Exposure of purified KGDH to arsenite displayed inhibition of enzyme activity. Comparison of arsenite concentration to enzyme inhibition has and will determine the relative toxicity and reactivity within the cell. It also determined a mechanism for oxidative stress due to arsenite exposure.

Methods and Materials:

Purified Enzyme Assay

KGDH purified from porcine heart was obtain from Sigma in a solution containing 50% glycerol, 10 mg/mL bovine serum albumin, 30% sucrose, 2.5 mM EDTA, 2.5 mM b-mercaptoethanol, 0.5% Triton X-100, 0.005% sodium azide, and 25 mM potassium phosphate, (pH 6.8). To measure enzyme activity, 1.02U of enzyme were centrifuged for 30 minutes at 14,000g and 4^o C in Microcon YM –30 Centrifugal Filter Devices. The enzyme was placed in 1.8mL 120 mM KCl, 5.0 mM KH₂PO₄, and 5.0 mM MOPS at pH 7.25 (Buffer 1). 15 mL of Enzyme/Buffer 1 solution were added to 200 mM CoASH, 0.2 mM TPP, 10 mM CaCl₂, and 0.5 mM a-ketoglutarate in buffer 1. As(III) was added accordingly to achieve the desired concentration of As(III) and a final volume of 200mL after the addition of NAD⁺. The solution was incubated for 30 min. at 30^o C. 0.5 mM NAD⁺ was added and NADH formation was recorded at 340nm in a 96 well spectrophotometer.

Dithio-(bis)nitrobenzoic Acid (DTNB) Assay

The reduction of DTNB by lipoic acid measured at 30^o C by an increase in absorbance at 412 nm. Prior to measurement, it was necessary to remove the b-mercaptoethanol from the KGDH preparation. This was done by centrifugation of 1.02U of enzyme in Microcon YM –30 Centrifugal Filter Devices at 14,000g and 4^o C for 30 min. Remaining enzyme was added to Buffer 1 containing 100 mM NADH creating a final volume of 2 mL. Indicated amounts of arsenite were added and the mixture incubated for 30 min. 100 mM NADH and 0.5 mM DTNB were then added and the DTNB reduction was measured at 412 nm.

Western Blot & Coomasie Stain/Mass Spectroscopy Preparation

102 mU of KGDH was mixed with 80 mL of 1.0M Tris (pH 6.8), and centrifuged for 10 minutes at 14,000 g and 4^o C in Microcon YM –30 Centrifugal Filters. 80 mL of Tris were added and the solution gently shaken for 30 sec. It was centrifuged again for 10 min. at 14,000 g and 4^o C. 80 mL of 1.0 M Tris (pH 6.8) was added for a final time and gently mixed for 30 sec. The filter was inverted and the protein recovered in another tube at 1000 g for 3 minutes. 5 mL of enzyme/Tris solution was added to Buffer 1 and indicated amounts of arsenite. These were incubated for 30 min at 30^o C. Sequencing grade purified trypsin (0.033mg ) (Promega) was added and the solution incubated for another 30 min at 30^o C to separate the KGDH subunits. 1mg of Soybean trypsin inhibitor was added and the solution was again incubated for 30 min at 30^oC. Samples were then run on a 6% PAGE gel for ~80 minutes at 150V. The gel was then cut in half. One half was developed by western blot analysis as indicated below, the other half was stained with Coomasie stain overnight. The desired bands were cut out of the gel and a trypsin in-gel digest was performed allowing analysis by Mass Spectroscopy as described below.

Western Blot

The gel was transferred onto a nitrocellulose membrane at 1A for 48min. at 4^o C. The membrane was washed 3 x 5 min. with Tris buffered saline containing Tween-20 (TBS-T). 1:1000 anti-lipoic acid rabbit polyconal antibody[ii] was added and the membrane rocked at 30^o C for 1 hr. The membrane was washed 3 x 5 min with TBS-T, 1:10000 goat anti-rabbit antibody was added, and the membrane was rocked for an additional 35 min. at 30^o C. The membrane was then washed 3 x 5 min. with TBS-T, dried, and 1:1 ECL solution was applied for 1 min. The membrane was then dried, and the presence of enzyme measured by exposure of light sensitive film (Kodak) in a dark room.

Mass Spectroscopy

The gel was destained with 10% MeOH and 10% acetic acid for 3 hr. Eppendorf tubes were prewashed with 0.1% trifluoroacetic acid / 60% CH₃CN. The KGDH E2 subunit that corresponded to the a-lipoic acid antibody band identifited by Western blot analysis was cut out of the gel and put into the prewashed eppendorf tubes with 250 mL 50% H₂O / 50% CH₃CN and washed for 5 min. The wash solution was removed, 250 mL 50% CH₃CN / 50 mM NH₄HCO₃ was added, and the gel pieces washed for 30 min. The wash was again removed and 250 mL 50% CH₃CN / 10 mM NH₄CO₃ was added. The 30 min. wash and removal of the wash was performed one final time, and gel pieces were speedvaced to complete dryness. 0.1 mg sequencing grade purified trypsin (Promega) was added per 15 mm³ of gel to break the KGDH subunits into small peptide sequences. The gel pieces sat for 10 min. and then 10 mM NH₄HCO₃ was added. This was then incubated at 37^o C for 24 hours. 200 mL 0.1% TFA, 60% CH₃CN was added and the tube were shaken for 1 hr. The wash was collected and stored. The wash was performed again, and the washes were pooled and speedvaced to dryness. The tryptic fragments were then resuspended in 500 mL of 0.05% TFA / 5% CH₃CN and IES-TOF mass spectroscopy was performed.[iii]

Results

Effects of arsenite on KGDH activity. Incubation of purified enzyme with arsenite showed loss of enzyme activity. As the amount of arsenite was increased, enzyme activity decreased (Figure 1). Inhibition of KGDH was seen as low as 50ppb As(III) and enzyme activity was completely inhibited at 5ppm.

Figure 1: Loss of NADH formation due to inhibition of KGDH by As(III)

To determine the site of arsenite binding and the type of inhibition, the V_max of the substrate for the E1 complex, a-ketoglutarate, was examined at constant arsenite concentrations, but the almost equal slopes of the a-ketoglutarate variations (Figure

2) indicate that non-competitive inhibition exists by arsenite on the E1 complex of KGDH. This confirms our hypothesis that As(III) does not bind to the E1 subunit of KGDH.

Figure 2: KGDH activity with varying amounts of a-ketoglutarate, the E1 subunit substrate

DTNB Assay: To investigate whether As(III) binds to the lipoic acid modification on the E2 subunit, reduction of DTNB by the E2 subunit was measured with increasing As(III) concentration. As As(III) binds to the lipoic acid cofactor on the E2 subunit, it causes the loss of lipoic acid reduction ability. This loss of reduction capabilities was monitored through loss of DTNB reduction.

Figure 3: Measurement of DTNB reduction by lipoic acid on the E2 subunit with varying amounts of As(III) at 412nm.

As As(III) concentration was increased, DTNB reduction decreased. As(III) blocked the reduction site of lipoic acid preventing bond formation between DTNB and the lipoic acid cofactor (Figure 3).

Time Dependent KGDH Activity Assay: To determine the strength of the bond between lipoic acid and arsenite, and whether or not arsenite binding is reversible, time dependence of KGDH activity inhibition was measured at an arsenite concentration of 250 ppb. This concentration was chosen because it exhibited only 25% inhibition after 30 min. of incubation (Figure 4).

Figure 4: Incubation of As(III) with varying amounts of time indicating irreversibility of As(III) binding at the E2 subunit.

It should be noted that the last three data points show a leveling trend. This indicates the incubation time required for complete arsenite binding to lipoic acid. The three points displaying a level trend also indicate that arsenite binding is irreversible. Once arsenite binds to lipoic acid, the inhibition is permanent.

Western blot / Coomasie stain: In order to establish an arsenite binding site we performed ESI-TOF mass spectroscopy on tryptic digests of the E2 subunit. A peptic/tryptic fragment with bound arsenite should display a shift of 75 mass units, the mass of an arsenic atom. Western blot was used in conjunction with a Coomasie stain and an in gel digest to be able to perform mass spectroscopy and determine lipoic acid as the reactionary site for arsenite. Due to the small size of the arsenic atom, the recognition of lipoic acid by the anti-lipoic acid antibody was not altered. This enabled us to determine the exact location of the E2 subunit in the gel using Western blot techniques (Figure 5). With the exact location of the E2 subunit known, it could be cut from the gel, digested, and mass spectroscopy performed.

Figure 5: Identification of lipoic acid and the E2 subunit of KGDH through Western blot allowing removal of the corresponding gel band for tryptic digest and mass spectroscopy.

To help stabilize KGDH, it was supplied in a solution containing bovine serum albumin (BSA). The BSA band is readily visible at 69kDa below the KGDH E2 band. Despite purification, the tryptic BSA fragments have been identified with mass spectroscopy, as well as unidentified fragments believed to be KGDH.

Future Plans

With the discovery of KGDH inhibition by As(III), it is important to determine the exact location and means of this inhibition. As(III) was not found to inhibit KGDH at the E1 subunit. Loss of KGDH reduction ability strongly indicates inhibition of the E2 subunit but is not complete confirmation. Kinetic studies with substrates for the other two subunits need to be performed. Activity assays should also be performed with 100 min. As(III) incubation, enabling As(III) to perform its full inhibition of lipoic acid as discovered in the time dependent activity assay. In addition to the activity assays, further Western blot analysis will be conducted using larger arsenic containing organic ligands. It is clear that the binding of arsenite is insufficient to lose antibody recognition. This is not consistent with studies by Sweda et al.,ii who observed binding loss modification of lipoic acid by aliphatic (C-9) carbon chain cofactor. This should lead to loss of the lipoic acid recognition by the anti-lipoic acid antibody. Radioactive arsenite will also be used in a Western blot to indicate arsenite binding to the E2 subunit. Finally, in preparation for mass spectroscopy, sequencing grade purified chymotrypsin will be used as a protease for the in gel digest instead of trypsin due to the trypsin’s cleavage at lysine residues; as lipoic acid is attached to a lysine. The modification of a lysine residue is a confounding factor for the post-analysis software.

Acknowledgements

This work was supported in part by University of Montana IBS-CORE, University of Montana Department of Chemistry Lien Fellowship, National Institute of Environmental Heath Sciences (Grant ES10437), and the Murdock Foundation (Grant M25548). The author of this paper would also like to thank Dr. Doug Williamson of the School of Pharmacy mass spectroscopy facility at the University of Montana for performing and analyzing mass spectroscopy data, and Dr. Brooke Martin for acting as a mentor during the duration of this research.